In 1993, Langer and Vacanti et al. estimated the number of bone repair procedures performed in the United States at over 800,000 per year (Science 1993 260(5110):920-926). Today, skeletal reconstruction has become an increasingly common and important procedure for the orthopaedic surgeon. Conventional approaches in bone repair have involved biological grafts such as autogenous bone or autografts, allogenic bone or allografts and xenografts (Burwell, R. G. History of bone grafting and bone substitutes with special reference to osteogenic induction, in Bon Grafts, Derivatives and Substitutes., M. R. Urist and R. G. Burwell, Editors. 1994, Butterworth-Heinemann Ltd.: Oxford. p. 3). Currently, autograft is the preferred biological graft most often utilized in the clinical setting, having success rates as high as 80-90% and no risk of immune rejection or disease transfer (Cook et al. J. Bone Joint Surg. Am. 1994 76(6):827). However, due to limited availability of autografts and risks of donor site morbidity, alternative approaches to bone repair have been sought.
Numerous tissue engineering solutions have been proposed to address the need for new bone graft substitutes.
One potentially successful repair solution seeks to mimic the success of autografts by removing cells from the patient by biopsy and growing sufficient quantities of mineralized tissue in vitro on implantable, three-dimensional scaffolds for use as a functionally equivalent autogenous bone tissue. In this way, an ideal bony repair environment is created by reproducing the intrinsic properties of autogenous bone material, which include: a porous, three-dimensional architecture allowing osteoblast, osteoprogenitor cell migration and graft re-vascularization; the ability to be incorporated into the surrounding host bone and to continue the normal bone remodeling processes; and the delivery of bone forming cells and osteogenic growth factors to accelerate healing and differentiation of local osteoprogenitor cells (Burwell, R. G. History of bone grafting and bone substitutes with special reference to osteogenic induction, in Bone Grafts, Derivatives and Substitutes., M. R. Urist and R. G. Burwell, Editors. 1994, Butterworth-Heinemann Ltd.: 5 Oxford, p. 3; Gadzag et al. J. Amer. Acad. Ortho. Surg. 1995 3 (1): 1).
Biodegradable scaffolds for in vitro bone engineering, which possess a suitable three-dimensional environment for the cell function together with the capacity for gradual resorption and replacement by host bone tissue have also been described. See, e.g. Casse-bette et al. Calcified Tissue International 1990 46(1):46-56; Masi et al. Calcified Tissue International 1992 51(3):202-212; Rattner et al. In Vitro Cellular & Developmental Biology-Animal 1997 33(10):757-762; Mizuno et al. Bone 1997 20(2):101-107; El-Ghannam et al. J. Biomed. Mater. Res. 1995 29(3):359-370; Ducheyne et al. J. Cell. Biochem. 1994 56(2):162-167; Ishaug et al. J. Biomed. Mater. Res. 1997 36(1):17-28; Ishuag-Riley et al. Biomaterials 20 1998 19(15):1405-1412; Goldstein et al. Tissue Engineering 1999 5 (5): 421-433; Devin et al. J. Biomater. Science-Polymer Edition 1996 7(8):661-669; Laurencin et al. Bone 1996 19(1):593-599; Thomson et al. Biomaterials 1998 19(21):1935-1943; and Laurencin et al. J. Biomed. Mater. 2 5 Res. 1996 30(2):133-138. This three-dimensional matrix milieu provides the necessary microenvironment for cell-cell and cell-matrix interaction, and is sufficient for the production of limited amounts of mineralized bone matrix in static culture. To demonstrate clinical feasibility of tissue engineered bone and to sufficiently match the intrinsic properties of autogenous bone graft material, however, rapid mineralization of osteoid tissue grown in vitro must be achieved. In the above-described three-dimensional matrices, nonhomogeneous cell seeding 35 confines cell density to the near surface of the scaffold and mineralized tissue formation is limited by inadequate diffusion of oxygen, nutrients, and waste.
Using porous polylactic glycolic acid (PLAGA) foams with pore sizes ranging from 150 to 710 μm, Ishaug-Riley et al. (Biomaterials 1998 19(15):1405-1412) have observed a limit to osseous tissue ingrowth and mineralization in a static culture environment of about 200 μm. While it is possible that structures with larger pores would facilitate greater diffusion, important cell-cell interactions and scaffold mechanical integrity could be compromised.
Formation of three-dimensional assemblies for culturing of various cell types in a rotating bioreactor have been described. See e.g. Goldstein et al. Tissue Engineering 1999 5(5):421-433; Granet et al. Cell Eng. 1998 36(4):513-519; Klement et al. J. Cellular Biochem. 1993 51(3):252-256; Qui et al. Tissue Engineering 1998 4(1):19-34; Lewis et al. J. Cellular Biochem. 1993 51(3):265-273; Becker et al. J. Cellular Biochem. 1993 51(3):283-289; and Prewett et al. J. Tissue Culture Methods 1993 15:29-36. Using such assemblies, it has been shown that osteoblast-like MC3T3 cells form cell aggregates when grown on non-degradable microspheres and produce collagen fibrils in the matrix between microspheres (Klement et al. J. Cellular Biochem. 1993 51(3):252-256). Also, rat stromal cells cultured for 2 weeks on cytodex-3 beads formed aggregates, began synthesizing mineralized matrix and showed elevated expression of type I collagen and osteopontin (Qui et al. Tissue Engineering 1998 4(1):19-34). However, when microspheres with greater density than the surrounding medium are placed in a rotating bioreactor, centrifugal force induces heavier-than-water microspheres to move outward and collide with the bioreactor wall. These collisions induce cell damage and are a confounding variable in tissue engineering.
In the present invention, lighter than or light as water, biocompatible, biodegradable microcarriers and scaffolds comprising these microcarriers are used in a three-dimensional culturing method for the growth of mineralized tissues in vitro in a rotating bioreactor. The combination of three-dimensionality and fluid flow of the present invention circumvents limitations associated with static three-dimensional culturing methods, eliminates confounding wall collisions, and increases the rate and extent of mineralized tissue formation in the rotating bioreactor. Scaffolds prepared in accordance with the present invention exhibit controllable and quantifiable motion in a bioreactor environment, thereby enhancing fluid transport throughout the scaffold. As demonstrated herein, scaffolds produced in accordance with the present invention support cell attachment, growth, and phenotypic expression over short-term culture ultimately resulting in enhanced synthesis of mineralized bone graft quality tissue.